Our results indicate an OT specific activation of PKR that

Our results indicate an OT-specific activation of PKR that inactivates eIF2a and may, by this means, reduce protein translation by utilizing only select sensors and mediators of the UPR (i.e., avoiding significant PERK activation). Interferon is a PKR activator [32] that temporarily subdues cellular and viral mRNA translation without killing the host cell [33]. Interferon transcription can be induced by XBP1s [39]; Interferon may thus allow for eIF2a inactivation via PKR after OT treatment. Our observation of minimal PERK activation agrees with Martinon et al. [39] who demonstrated that LPS-TLR2/4 stimulation of macrophages repressed PERK and IRE1a activation in combination with tunicamycin, which by itself activated these ER-sensors and who suggest that bacterial endotoxin-induced ER-stress specifically utilizes IRE1a and XBP1s, but not ATF6 and PERK. However, this study only detected XBP1 splicing with 3h LPS stimulation (vs. our 30min). The three main sensors of ER stress IRE1, PERK and ATF6 can be activated in concert, especially when massive misfolding results from stimuli such as UV, oxidative stress, and tunicamycin. During such toxic stimulation, one may look for protein ap1 when the likelihood to restore homeostasis is low. There are, however, physiological situations where the rate of cargo accumulation in the ER is less acute, and the UPR runs in phases, out-of and back-into homeostasis [17] in a steady rhythm. This is physiological to the extent that ER stress/UPR cycling has been described as a local tissue circadian clock [40]. In this case BiP responses may be elicited by IRE1 activation of XBP1s without involvement of PERK and ATF6 [17] and waves of protein entering the ER are responsible for increased demand for BiP rather than pathological aggregates. Therefore, the ER stress module of selective IRE1/XBP1/BiP activation by OT, that excludes strong PERK activity, could be viewed as a physiological advantage [41]. Note that XBP1 has more than one function, for example, it is required for the cellular secretory machinery in the cytoplasm [42] and regulation of lipid synthesis [43]. Thus the selective activation of XBP1 by OT may be beneficial. We show that OT selectively affects ER stress markers (summarized in Table 1), excluding PERK, however, the direct downstream signaling targets of OT/OTR remain to be elucidated. Our findings resonate with a recent study demonstrating increased pIRE1a, XBP1s and BiP in uterine myocytes derived from women in active labor requiring cesarean section, and thus subject to OT-mediated uterine contractions [44]. However, this myocyte study did not examine PERK or eIF2a, nor the role of OT; further, it utilized stimulation with 10,000ng/ml LPS for 20h; these parameters represent a 25-fold increase in LPS and 40-fold longer stimulation duration from our studies [44]. OT may protect uterine myocytes from apoptosis during contractions by reducing protein translation selectively via pIRE1a and XBP1s with repression of pPERK and CHOP, and inactivation of eIF2a (by PKR). Low BiP response to LPS suggests that LPS fails to induce ER stress. To explain this phenomenon we looked at the effect of LPS on the abundance of A20, which increased 90min (actually, 120min, if we include the 30min of OT, see Fig. 8) after LPS stimulation. A20 is involved in the downregulation of the inflammatory response to LPS by a negative feedback loop [35]. OT only partially suppressed increased A20, which supports that A20 protected the cells after LPS stimulation by disrupting LPS signaling within the temporal and dose parameters of these experiments. LPS-stimulated NF-κB transcriptional programs have a positive anabolic effect upon multiple genes [31], some of which negatively regulate NF-κB such as A20 [45], [46] and increase cellular tolerance to LPS [35] thereby promoting apoptosis. In our study OT only partially inhibited the increases in A20 stimulated by 90min LPS treatment. As increases in A20 would attenuate the impact of LPS and potentially thwart apoptosis, further studies should explore whether other doses or treatment durations of LPS and OT (e.g., [45]) will reveal a more significant impact of OT upon A20. Attenuation of a cell\'s ER-stress sensor PERK can also be anti-apoptotic; PERK activation (and increased CHOP) initiates apoptosis in intestinal stem cells stimulated with LPS/TLR3 [47]. This model of stem cell apoptosis is implicated in necrotizing enterocolitis (NEC), a common and severe complication observed in formula-fed, premature infants, and shown not to require IRE1a/XBP1 signaling [47] for which ER-stress-evoked autophagy has been demonstrated to be critical for cell death [48], [49].